Serologic and molecular studies to identify neonatal alloimmune neutropenia in a cohort of 10,000 neonates.

Neonatal alloimmune neutropenia (NAIN) is caused by maternal alloimmunisation to fetal human neutrophil antigens (HNAs). This study investigated maternal HNA/HLA alloantibodies involved with NAIN and identified the frequency of NAIN in Brazilian neonates. Neonatal neutropenia (neutrophil count < 1.5 × 109 /L) was investigated in samples from 10,000 unselected neonates, resulting in 88 neutropenic newborns (NBs) and their 83 mothers. Genotyping was performed by PCR-SSP (HNA-1/-4) and PCR-RFLP (HNA-3/-5). Serologic studies were performed by GAT (granulocyte agglutination test), Flow-WIFT (white blood cells immunofluorescence test) and LABScreen-Multi-HNA-Kit (OneLambda®) (LSM). Neonatal neutropenia was identified in 88/10,000 (0·9%) NBs. Genotyping revealed 60·2% maternal-fetal HNA incompatibilities (31·8% for HNA-1; 14·8% for HNA-3; 15·9% for HNA-4; 21·6% for HNA-5). Serologic studies revealed 37·3% of mothers with positive results with at least one technique. The detected anti-HNA specificities were confirmed in eight positive cases related to HNA-1/-3 systems. In cases with maternal-fetal HNA-4/-5 incompatibility, no specific neutrophil alloantibodies were found but anti-HLA I/II were present. Anti-HNA-2 was not identified. This is a large Brazilian study which involved the investigation of antibodies against all five HNA systems in neutropenia cases and showed a frequency of NAIN in 8/10,000 neonates. Among the HNA antibodies identified, we highlight the anti-HNA-1d and anti-HNA-3b, antibodies unusual in alloimmunised women, and rarely related to NAIN cases.

Antibodies against human neutrophil antigens in non-transfused women with red blood cell alloimmunisation induced by pregnancy.

BACKGROUND: Alloantibodies against human neutrophil antigens (HNA) resulting from allogeneic exposure may be associated with transfusion-related acute lung injury and immune neutropenia. Understanding the risk factors for the formation of such antibodies could have a great impact on the adoption of measures to prevent potentially fatal transfusion reactions. The aim of the study was to determine the prevalence of anti-HNA alloantibodies in non-transfused pregnant women with and without red blood cell (RBC) alloantibodies. MATERIALS AND METHODS: HNA alloantibodies were investigated in blood samples from 147 pregnant women with RBC alloimmunisation induced by pregnancy as the only allogeneic stimulus (group 1). The control group (group 2) consisted of 563 women with at least one pregnancy without RBC alloimmunisation. Both groups were investigated for the presence and identity of HNA alloantibodies using granulocyte agglutination tests, white blood cell immunofluorescence testing, and the bead-based LABScreen Multi Kit. Genotyping was performed to confirm the specificity of the HNA alloantibodies. RESULTS: Group 1 women had a statistically higher number of HNA alloantibodies compared to group 2 women (9/147 [6.1%] vs 9/563 [1.6%]; p=0.005, OR=4.01; 95% CI 1.5-10.3). Considering only multiparous women, there was a higher statistical significance for the difference in the presence of HNA alloantibodies between the two groups (7/82 [8.5%] vs 9/493 [1.8%]; p=0.002, OR=5.02; 95% CI 1.8-13.9). DISCUSSION: Our data show that RBC alloimmunisation is significantly associated with the development of anti-HNA alloantibodies, corroborating the hypothesis that some individuals are better immune responders and react strongly to allogeneic exposure. The presence of RBC alloantibodies can, therefore, facilitate the identification of individuals with a higher risk of alloimmunisation to antigens from other cells, also acting as a tool to avoid potentially fatal transfusion reactions.

Effects of Immunosenescence on the Lower Expression of Surface Molecules in Neutrophils and Lymphocytes.

BACKGROUND: Immunosenescence is a remodeling of the immune system, caused by aging, with changes in the function of neutrophils, lymphocytes, and Treg cells. OBJECTIVE: This study aimed to evaluate the expression of molecules CD11b, CD16 and CD64 (neutrophils), CD154 (T lymphocytes), CD40 (B lymphocytes), and to quantitatively analyze the Treg cell subpopulation. METHODS: 49 elderlies (≥60 years) and 49 adults (≤35 years) were studied. Flow cytometry was used to analyze the expression of surface molecules and the subpopulation of Treg cells, and the results between the groups were compared statistically by the t-test. RESULTS: There was a decreased significance in the expression of CD11b and CD40 in the elderly. CONCLUSION: Decreased CD11b expression can result in susceptibility to infectious diseases, and impairment of phagocytic capacity. Decreased CD40 expression can result in a decline in B lymphocyte activation. The other molecule studied presented alterations not significant, but compatible with the immunological changes in aging.

Impact of using different laboratory assays to detect human leukocyte antigen antibodies in female blood donors.

BACKGROUND: HLA antibodies passively transferred to transfused recipients may cause transfusion reactions such as transfusion-related acute lung injury (TRALI), but in many of the reported TRALI incidents, no white blood cell antibodies have been identified. We investigated whether a higher number of anti-HLA would be detected in donor's plasma by using a method with potential higher sensitivity rate. STUDY DESIGN AND METHODS: Sera from 300 previously pregnant female blood donors were screened for anti-HLA using a solid-phase mixed-antigen assay (enzyme-linked immunosorbent assay [ELISA]). Samples from 60 women with three or more pregnancies with a negative ELISA were further tested using microbead-flow assays (LABScreen mixed, panel-reactive antibodies [PRA], and single antigen). RESULTS: Anti-HLA Class I and/or Class II were detected by ELISA in 26.7% (80/300) of all women and in 37.0% (37/100) of women with three or more pregnancies. The LABScreen assays detected additional anti-HLA specificities (44 Class I and 17 Class II) in 28.3% (17/60) of ELISA-negative donors with three or more pregnancies. HLA antibodies were detected in 8.3% (5/60), 18.3% (11/60), and 21.7% (13/60) of ELISA-negative women by LABScreen mixed, PRA, or single antigen, respectively. CONCLUSION: Our data showed that the microbead-flow detected more HLA antibodies than ELISA, but the clinical significance of these antibodies is currently unknown. Detecting anti-HLA is useful for donor management and could contribute to the decision to definitively defer blood donors involved in TRALI incidents. However, further studies are necessary to better determinate the relative risk of TRALI induced by anti-HLA detected only by techniques with higher sensitivity rate.